Atherosclerosis is the chronic inflammatory response that leads to stenosed blood vessels. Highly organized and multilaminate in structure, native blood vessels have complex 3D spatial organization with anisotropic contractile properties. Although cell sheet engineering technologies have been devised to allow recovery of aligned cells without damage, native mechanical properties using mechanical conditioning may also be replicated. A device was created using an elastic membrane that can mechanically condition cells for cellular alignment and secretion of proteins and extracellular matrix (ECM), and then allow for a thermoresponsive spontaneous, nondamaging detachment of those cells and ECM fully intact for a scaffold-free cellular implant. We aim to engineer functional tissue engineered blood vessels by creating cell sheet monolayers with controlled ECM growth, which are subsequently stacked and rolled to create a durable blood vessel for implantation. The central hypothesis of this proposal is that layered sheets of pre-conditioned vascular smooth muscle cells results in mechanical architecture and function that more closely mimics native blood vessel mechanics than layered cell sheets without prior conditioning. Aim 1: Nonconditioned or anisotropically conditioned vascular smooth muscle cell sheets will cause cellular alignment comparable to current cell sheet engineering technologies. Cells will be mechanically conditioned at 10% cyclic elongation strain. Immunostaining will be used to identify actin fibers for cellular orientation analysis. We can also measure cell sheet shrinkage after removal from our thermoresponsive surfaces that allow nondamaging cell detachment. Aim 2: Cellular sheets of vascular smooth muscle cells secrete higher protein and extracellular matrix content as a result of mechanical conditioning. Conditioned and nonconditioned cells will be evaluated qualitatively by immunohistochemistry and quantitatively by RT-PCR and Western analysis, to determine RNA and protein expression levels of vascular proteins. Biochemical assays will also be performed to obtain a quantitative ECM profile. Aim 3: Conditioned cell sheets have architecture and mechanical properties more similar to native blood vessels than nonconditioned cell sheets. We will examine individual layers and layered constructs using transmission electron microscopy (TEM) to examine architecture. Mechanical testing using an Instron machine can measure for elasticity and tensile strength.